The binding of fluorescein-labeled protein synthesis initiation factor 2 to Escherichia coli 30 S ribosomal subunits determined by fluorescence polarization.
The binding of initiation factor IF2 to 30 S ribosomal subunits was studied quantitatively by steady state fluorescence polarization techniques. Covalent fluorescent derivatives of IF2 were prepared by treating the pure factor with fluorescein isothiocyanate. Derivatives (F-IF2) containing about 1 fluorescein molecule/protein bound to 30 S ribosomal subunits as measured by sucrose gradient centrifugation; however, their activity is slightly impaired in assays for stimulation of formylmethionyl-tRNA binding to 70 S ribosomes. Upon addition of 30 S ribosomal subunits to F-IF2 solutions, a significant increase in fluorescence polarization results, but no change in quantum yield or fluorescence lifetime is detected. Fluorescence polarization values were measured at different F-IF2 and 30 S ribosomal subunit concentrations and the equilibrium association constant and number of ribosomal binding sites were calculated. The association constant for F-IF2 in buffer containing 100 mM NH4Cl and 10 mM Mg acetate is 1.5 +/- 0.2 X 10(7) M-1; the number of binding sites is 0.9 +/- 0.2. Competition of F-IF2 with IF2 showed that the nonderivatized factor binds somewhat more tightly than F-IF2, with an association constant of 2.7 X 10(7) M-1. The binding of F-IF2 is strongly inhibited by raising the NH4Cl concentration from 20 mM to 150 mM, but is rather insensitive to Mg acetate concentrations between 2.5 and 10 mM. Addition of initiation factors IF1 and IF3 causes a 3-fold increase in the association constant for F-If2 binding to 30 S subunits.
D000643:Ammonium Chloride / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D007700:Kinetics; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism*; D011485:Protein Binding; D012270:Ribosomes / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013861:Thiocyanates
D005452:Fluoresceins; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D013861:Thiocyanates; D000643:Ammonium Chloride; D016650:Fluorescein-5-isothiocyanate